1. Field of the Invention
The present invention is broadly concerned with an improved embryo collection device of the type used for extracting and identifying mammalian embryos from an embryo-containing irrigant fluid. More particularly, it is concerned with such an embryo collection device which in preferred forms includes an outer vessel provided with a light-transmitting base permitting the collection device to be situated within a microscope in order to allow direct identification and counting of extracted embryos without the need for transfer of such embryos to a counting dish. The outer vessel advantageously includes an outlet port spaced a distance above the bottom wall for conveying filtered irrigant fluid from the vessel device while allowing retention of sufficient fluid within the collection device to prevent the filter and collected embryos from drying out. A specialized fluid inlet nipple is also provided which discharges incoming irrigant fluid at a level below the minimum fluid level maintained within the vessel, thereby avoiding splashing and formation of foam.
2. Description of the Prior Art
Mammalian (e.g., bovine) embryos are routinely removed from female donor animals by a process which involves flushing of the donor animal uterus with an irrigant fluid. The embryo-containing irrigant fluid is then collected, and steps are taken to extract the embryos from the relatively large volume of collected irrigant fluid. It has been known in the past to collect embryo-containing fluid in a container, while attempting to keep the irrigant fluid moving. As a part of this procedure, the irrigant was decanted or otherwise drained from the container, and the embryos were then transferred to a petri or other counting dish for examination. This approach is inherently time-consuming and necessitates handling of the embryos between the settling container and the counting dish.
U.S. Pat. No. 4,563,172 describes an embryo collection device having an intermediate collection chamber equipped with a filter, together with an upper cover member provided with an irrigant fluid inlet, and a funnel-shaped receipt chamber beneath the filter with a discharge aperture at the bottom of the receipt chamber. One significant problem with this collection device is that the irrigant fluid inlet directs the incoming fluid vertically downwardly in an unimpeded fashion for a substantial free-fall distance within the device. This inevitably creates a foamy "head" on the fluid within the device due to the serum component in the irrigant, which often requires aspiration of the head in order to permit visualization of the microscopic ova which are easily obscured by the foamy bubbles. The aspirated head must be retained and carefully inspected though, inasmuch as valuable embryos can be carried over in this fraction.
The presence of foam in the search dish is undesirable since it obscures vision and therefore greatly slows the microscope search process. Embryos also stick to bubbles and at that point are very difficult to locate and extract. Most technicians aspirate the foam head off with a syringe immediately prior to searching and do not wait 10-20 minutes for the foam head to dissolve. The drawback to aspiration is that embryos attached to bubbles will unknowingly be removed in the syringe
As described in the '172 patent, in the use of the collection device it is important to maintain a level of fluid in the collection chamber sufficient to keep the embryos in suspension and prevent the embryos from settling against the filter; otherwise, the embryos may be unacceptably damaged. This mode of operation is also necessitated by virtue of the straight-through vertical design of the irrigant fluid inlet and the consequent likelihood of embryo damage if an insufficient volume of fluid is present in the device. Moreover, inasmuch as the patented collection device includes a funnel-shaped receipt chamber beneath the filter, it is necessary to transfer the collected embryos to a separate counting dish for detailed examination, and this constitutes another undesirable handling step.
If, contrary to the teachings of the '172 patent, collected embryos actually settle out or otherwise come into contact with the filter, the device must be repeatedly rinsed and swirled in an effort to resuspend all of the embryos for transfer examination and counting. This problem is particularly acute given the foaming tendency of the irrigant fluid and because the fluid includes uterine endometrial and mucous membrane debris which can entrap embryos. Therefore, it is often very difficult to insure that all embryos are properly transferred when using the device of the '172 patent.
A more recent embryo collection device is described in U.S. Pat. No. 4,781,706, which illustrates a vessel with a bottom wall and an upstanding sidewall presenting a filter opening above the bottom wall; an embryo filter is attached to the sidewall opening. A cover member having an irrigant fluid inlet is also provided, which permits incoming embryo-containing fluid to be passed into the vessel for filtration. As with the '172 patent, the irrigant fluid inlet directs the incoming fluid vertically downwardly in an unimpeded fashion for a substantial free-fall distance within the device, creating a foamy "head" as described above. Another significant problem with the collection device provided by the '706 patent is that after filtration is complete, only a low level of irrigant fluid remains within the vessel, this level being dictated by the height of the filter above the vessel bottom wall. As a consequence, however, the filter itself necessarily dries out because it is no longer exposed to irrigant fluid after filtration. This in turn means that valuable embryos can adhere to the filter, dry out and be lost.
Prior art embryo collection devices suffer from a number of other limitations. For example, entrance ports to prior art devices are straight male ports which require the user to wedge the end of the Y-junction tubing over and down the shaft, which can be difficult to accomplish on a cold day when the tubing is stiff. Another drawback to this prior art design is that after the embryos are collected, the user must separate the tubing from the device in order to transport the device to the microscope. This separation is accomplished by either pulling, twisting, or stretching the tubing until the friction is overcome. As a result, the tubing often pops loose suddenly from the nipple and violently jars the device and embryos therein. Or, one must take scissors and cut the tubing to separate it from the device.
Lastly, this attachment method is not as secure as one would like since in cold weather the tubing friction hold on the nipple is reduced since tubing pliability and "stickiness" is reduced in cold temperatures.
Another limitation of prior art collection devices is that they have lids which are connected to their lower collection vessels by a snap-on or friction attachment arrangement. Friction attachment is adequate if the practitioner has an assistant to hold the filter with "kid gloves" and the device is never dropped, bumped, or left to hang suspended by tubing over a bar on the cattle squeeze chute. However, all of the above situations occur routinely and therefore many practitioners use rubber bands to secure the lid to the base, or ask someone to hold the filter throughout the collection period. The lid is even more likely to disconnect from the base in cold weather. Cold weather causes frequent problems because most dairy and beef cattle are collected year-round, most exclusively on-farm in the barn.
All known self-draining, direct-examination devices are also plagued with the problem of leaking fluid onto the microscope stage. This occurs as the dish is moved up, down, and back and forth across the scope stage during the search process which causes fluid to splash either against or through the filter and onto the stage. To avoid this design flaw, the user must take a syringe and aspirate a substantial amount of fluid out of this "micro-pool" prior to placing the vessel onto the stage. Unfortunately, syringe aspiration in this limited fluid depth can unknowingly draw embryos up into the syringe.
The standard petri dish used most exclusively today for embryo isolation/extraction has several handicaps. In the standard petri dish, embryos sink in the fluid and come to rest on top of the horizontal bottom wall of the dish. As the technician moves the dish in an "S" pattern over the microscope stage during the search process, embryos roll and change location in the dish. Even when a gridded pattern is etched on the bottom, embryos roll around (shift position) in the bottom of the dish due to the fluid wave motion inevitably caused by dish movement over or across the scope stage.